Thrombin-free biological adhesive and use thereof as a medicament

ABSTRACT

The invention relates to a thrombin-free, fibrinogen-based biological adhesive for therapeutic use, which comprises factor Vila and a source of calcium ions. The invention also relates to the use of the biological adhesive as a medicament, in particular as a dressing for biological tissues, wounds or biomaterials.

The present invention relates to a fibrinogen-based biological adhesivewith an activated factor VII and a source of calcium ions.

By “biological adhesive” is meant a component capable of joining tissueelements (skin, bone, various organs) and in the same time providinghaemostasis of the damaged tissues, which may thus reinforce or completejoining of these tissues by suture(s).

Blood coagulation is achieved according to steps in cascade involvingdifferent proenzymes and procofactors present in blood which areconverted via proteolytic enzymes into their activated form. Thissuccession of coagulation steps or cascade is carried out according totwo coagulation systems, called the extrinsic coagulation route and theintrinsic coagulation route, leading to transformation of prothrombininto thrombin.

The extrinsic route involves the intervention of the factor VII presentin blood. However, the latter requires an activation (factor VIIa) forinitiating this coagulation cascade. The factor VIIa has low enzymaticactivity until it is complexed with tissue factors of phospholipidnature released after tissular damage. The thereby complexed factor VIIatransforms the factor X (FX) into the factor Xa (FXa) in the presence ofcalcium ions. The factor Xa in turn transforms prothrombin intothrombin, which activates the factor V (factor Va). Thrombin alsoactivates the factor XIII (factor XIIIa). Thrombin, in the presence ofcalcium, tissular factors, factor Va, acts on fibrinogen by transformingit into fibrin. The presence of factor XIIIa allows formation of a clotof fibrin with a solid and adherent meshed network which is graduallyand slowly resorbed with the setting up of the consolidation scartissue, at which the network is used as a frame. This cross-linkedfibrin is insoluble and cannot be attacked by fibrinolytic enzymes, atleast during the time for setting up the scar tissue.

The intrinsic coagulation route also involves the factor VIIa (FVIIa).This route comprises a cascade of reactions resulting in the activationof thrombin via the factor XII (FXII). The latter activates the factorXI (factor XIa-FXIa) which activates the factor IX (factor IXa-FIXa) inthe presence of phospholipid tissular factors (FT). The factor IXaparticipates in the activation of factor X into a factor Xa in thepresence of the factor VIIIa, of tissue factors and of calcium ions.This then leads to transformation of prothrombin into thrombin. Itshould be noted that the presence of the factor Xa or thrombin allowsactivation of the factor VII (factor VIIa).

It therefore appears that the factor XIIa plays a prominent role in themechanisms of intrinsic coagulation, resulting in the formation of ablood clot. It is used in treating haemophiliacs A having a circulatinginhibitor, i.e. a specific antibody which limits or prevents activationof the factor VIII (FVIII). The factor VIIa has the advantage of beingable to act locally in the presence of tissular factors released afterlesion of tissues causing haemorrhages, even in the absence of thefactor VIII or IX.

However, in the case of haemorrhagic tissular lesions caused by wounds,injuries or external surgical operations, tissue repair by suture is arequirement, notably for stopping haemorrhage by forming a fibrin clot,according to the mechanisms explained earlier. It may prove to benecessary to promote haemostasis of damaged tissues, notably in the caseof severe surgical operations.

As an example, Patent Application WO 93/06855 may be mentioned, whichdescribes a haemostatic composition, free of thrombin and of coagulationfactors, comprising the factor VIIa incorporated into a biologicallycompatible vehicle, the whole being applied on a haemorrhagic injury inorder to promote haemostasis and formation of a fibrin clot.

However, except for haemostasis of damaged tissues, the natural processfor healing tissue, either sutured or not, related to joining tissues inorder to avoid their impairment and accelerate the process, should alsobe accelerated, reinforced or even completed. This is notably possibleby locally applying a biological adhesive.

Biological adhesives consist of a mixture of circulating factors ofblood coagulation, notably of fibrinogenic plasma proteins and of thefactor XIII. They further require a supply of exogenous thrombin, anenzyme necessary for transforming fibrinogen into insoluble fibrin,which may be cross-linked by the factor XIII. Additional components arerequired for carrying out coagulation of fibrinogen, notably the calciumion, as CaCl₂, and possibly aprotinin, added for its anti-fibrinolyticproperties.

Such biological adhesives were in particular described in differentpublications and patents, such as EP 0 305 243, FR 2 448 900 and FR 2448 901, as well as their applications in miscellaneous clinicalsituations (“Fibrinkleber”, Proceedings of the Heidelberg Congress,1976, Ed. Schattauer).

Biological adhesives may also be used for binding biomaterials ofmiscellaneous nature (collagen, alginates, and polylactic acid) tobiological tissues in order to reinforce their mechanical propertieswhile waiting for consolidation by natural regrowth of the cells of theorganism, for example artificial skin.

Commercially available biological adhesives in reality appear as kitscomprising at least the four components mentioned above in a dry form,in which the proteins which may be coagulated by thrombin, fibrinogenand the factor XIII, should be isolated from thrombin, because theirassociation causes solidification in fibrin within a very short time, ofthe order of a few seconds after reconstitution in a liquid and mixing.This is the reason why biological adhesive kits are provided with atleast two components, i.e. comprising a batch based on fibrinogen and onfactor XIII on the one hand and a batch based on thrombin on the otherhand. The biological adhesive fulfils its functions indicated above byreconstitution and mixing of both batches, for example with syringes andneedles, and then by application on the tissue to be sutured.

However, such reconstitutions and mixtures are relatively complex and asource of possible errors, notably in the case of an emergency, giventhe fact that as soon as fibrinogen is in contact with thrombin as aliquid, in the presence of other components, fibrin is formed almostinstantaneously. Therefore the mixing of fibrinogen-thrombin may only beperformed just before application on the wound or operating area.Further, when a single device for dispensing biological adhesive isused, there is an actual risk of solidification of fibrin in the actualdevice, which may lead to blocking of the dispensing and applicationsystem.

Patent EP 0 850 650 B1 notably describes a pre-activated two-componentadhesive containing fibrinogen and at least one activated coagulationfactor, the activation of which does not depend on calcium ions,selected from the Factor XIIa, the Factor XIa, the Factor VIIa andkallicrein and it coagulates by simple addition of calcium ions, byforming fibrin. This adhesive is obtained by fractionating orconcentrating activated blood plasma from a single donor, in order toconcentrate fibrinogen while retaining the activated coagulation factorsrequired for allowing coagulation under the action of calcium ions. Suchan adhesive has the drawbacks of the aforementioned two-componentadhesives and of forming the fibrin clot upon mixingquasi-instantaneously.

Patent Application WO 02/055102 A1 describes a composition comprisingFVIIa and fibrinogen useful for limiting or stopping bleeding caused byhaemorrhages or internal or external injuries. These compositions areintended to be injected intravenously to the patient and they act at thehaemorrhage or the injury where all the factors and the calcium ions arepresent. The drawback related to the use of such a composition lies inthe fact that the latter interacts with the FTs which would have spreadout of the injury or the haemorrhage, through the blood stream forexample, and may cause with the calcium ions naturally present in blood,an untimely coagulation with the different factors present in the blood,causing clots out of the haemorrhagic area, with risks of thromboses.

The aforementioned drawbacks therefore pose problems in particularwithin the scope of surgical operations and/or urgency situations, whererapidity of the operating gesture is often a necessity. It is observedthat such drawbacks are capable of generating an inhomogeneous joiningof the tissues by formation of fibrin clusters before tissue adhesion,which may further be enhanced by retraction of the clot, finally leadingto an irregular scar, favourable to deshience (disjoining of both banksof a scar) and to post-operating secondary bleedings.

In order to remedy these drawbacks, the Applicant sought to develop abiological adhesive meeting a dual related goal. The first goal is toprovide a thrombin-free biological adhesive comprising plasmacoagulation factors without the risk of forming fibrin beforeapplication on the biological tissue. The second goal meets the need ofmaking such an adhesive available, also having increased capabilities ofhaemostasis of damaged tissues on the one hand and of joining tissuesleading to enhanced healing on the other hand.

The invention relates to a stable thrombin-free fibrinogen-basedsingle-compound liquid biological adhesive for therapeutic use,comprising the Factor VIIa and a source of calcium ions.

The Applicant found that it is possible to make a new medicamentavailable (a single-compound biological adhesive): a haemostasisadjuvant, a dressing, a heeling adjuvant or sealing-off agent byformation of emboli, for biological tissues, wounds or biomaterials,comprising both above plasma factors of interest and a source of calciumions.

By “single-compound”, is meant a joint use of the three components ofinterest, constitutive of the adhesive thereby forming a single liquidcompound, as opposed to two-component adhesives of the prior art.

These three components are therefore compatible with each other as aliquid, i.e. their combination, thereby forming a single-compoundsystem, does not cause quasi-instantaneous formation of fibrin. Thissingle-compound liquid adhesive is stable because these components maybe in the presence of each other in a liquid medium for a functional useof the latter and this even at least for 24 hours before this use,without the risk of premature triggering of the coagulation process.This stability of the adhesive is verified, to the extent that noformation of fibrin is observed during 24 hours of incubation at 37° C.

The biological adhesive of the invention fulfils its repairing functionfor tissues once it is applied at the wound or the cruentous tissuebecause thrombin is formed in situ by contact of the biological adhesiveon the wound or cruentous tissue.

Indeed, this in situ thrombin formation is provided by the Factor VIIawhich is therefore put into contact with all the plasma componentsallowing triggering and progression of the extrinsic or intrinsiccoagulation route. As explained earlier, its biological activity isdependent on the interaction with the endogenous tissular factors ofphospholipid nature. The activated Factor VII tissular factors complexin the presence of calcium ions transforms the factor X present in theplasma into an activated factor X, which transforms prothrombin intothrombin, an enzyme responsible for forming the clot by generatingfibrin in the presence of fibrinogen.

Formation of fibrin then occurs and consequently the adhesiveness of theobtained biological adhesive is reinforced, because fibrin then formsdirectly at the wound or tissue to be repaired by surgery, where thephospholipid cell components are exposed.

Although fibrinogen and calcium ions are already present naturally inblood, with their contribution, it is possible to reinforce and furtheraccelerate even more the tissue joining and haemostasis process by thebiological adhesive of the invention and this even when compared withthe adhesives of the prior art. This is a decisive advantage of thepresent adhesive.

The biological adhesive of the invention has many other advantages. Itsavailability avoids drawbacks in handling and preparing biologicaladhesives of the prior art, notably in the case of emergency situations,which improves its capabilities in tissue adhesive bonding, in terms ofdeshience and absence of secondary bleedings at the scar in formation.Typically, the joining process may be achieved effectively in less thanone minute, for example within 30 s.

As an example, the adhesive of the invention avoids the formation of“lumps” or clusters of fibrin visible at a wound which would beexplained by inhomogeneous setting of the adhesive, probably because ofthe fibrin formed before adhesion, the formed scar further having aclear line, as compared with an adhesive bond of equal duration obtainedfor example by using a two-component biological adhesive based on amixture of fibrinogen and containing the factor XIII, on the one hand,and a calcium thrombin mixture on the other hand, for which the aboveeffects are not observed.

It is simple to prepare and has increased time stability, insofar thatno formation of fibrin is observed for 24 hours of incubation at 37° C.As an example, it is therefore stable as a liquid for at least 24 hoursat room temperature, in particular 48 hours.

Within the scope of the invention, any fibrinogen and FVIIa of the priorart, preferably plasmatic, are suitable for making the adhesive, howeverprovided that they are compatible with the calcium ions of the therebyobtained adhesive as a liquid, i.e. their contacting does not causecoagulation of fibrinogen into fibrin, notably 24 hours before use.Indeed, both active ingredients, fibrinogen and FVIIa, should thereforebe lacking, in addition to a lack of residual thrombin (FIIa), incoagulation factors such that, in the presence of calcium ions, theintrinsic or extrinsic coagulation cascade is triggered. Suchcoagulation factors represent the factor II (FII) and FX, and preferablythe prothrombinic factors (factors II, VII, IX and X).

As an example, a maximum acceptable FII and FX content in fibrinogen isabout 0.1 IU/g of fibrinogen for each of them.

Preferably, fibrinogen and FVIIa of the adhesive do not stem frompre-activated plasma.

Fibrinogen, preferably virally secure, may be isolated from plasma byany method developed in the prior art. It may be the method described inEP 0 305 243 or even the one developed by the Applicant in PatentApplication FR 05 06640 according to which a fibrinogen concentrate maybe obtained. Transgenic fibrinogen may also be implemented.

The factor XIII, preferably virally secure, may be isolated from plasmaby any method developed in the prior art and it may advantageously formthe protein which accompanies fibrinogen during fractionation of plasma.In this case, application of the method described in Patent ApplicationFR 05 06640 is preferred. Transgenic factor XIII may also beimplemented.

The preparation of the factor VIIa, notably as a concentrate, preferablyvirally secure, is also known. As an example Patent EP 346 241 may bementioned. A recombinant (from Novo) or transgenic factor VIIa may alsobe used.

These active ingredients should however meet certain criteria of purity,as stated earlier, relating to the presence of notably certain otherplasma factors.

Preferably, the adhesive of the invention further comprises the factorXIII. An exogenous supply of factor XIII promotes cross-linking of thefibrin network, and consequently, its coagulating power and healingpotential.

The factor XIII, in particular as a concentrate, preferably virallysecure, may be isolated from plasma by any method developed in the priorart and it may advantageously form the protein which accompaniesfibrinogen during fractionation of the plasma. In this case, the methoddescribed in Patent Application FR 05 06640 is preferably applied.Transgenic factor XIII may also be implemented.

The adhesive of the invention may be made on an industrial scale bymaking two active ingredients available, fibrinogen and FVIIa, andcalcium ions.

Preferably, the present adhesive consists of the sole mixture offibrinogen, FVIIa and calcium ions. Such an adhesive exclusivelyconsisting of these three components has the advantage of only requiringtwo biologically active ingredients, which notably limits the costs forpreparing the adhesive on an industrial scale by the presence of aminimum but effective number of active components.

The constitutive components of the adhesive are present in effectiveamounts so that the adhesive may meet sought therapeutic goals.

The Applicant however observed that best results in terms of theaforementioned sought-after effects may be obtained when the contents ofboth active ingredients and of calcium ions are selected specifically.

Thus, the biological adhesive advantageously comprises fibrinogen in acontent comprised between 60 and 120 mg/mL, more preferably from 80 to100 mg/mL. Such a fibrinogen content in the adhesive provides at leastone also effective adhesive bonding, of damaged tissues for example,which is notably expressed by a tearing resistance of the adhesivebonding at least as satisfactory as the two-component adhesives of theprior art, such as larger than or equal to 125 g/cm².

It comprises in particular from 50 to 500 IU/mL, more preferably from 70to 300 IU/mL, especially between 80 and 120 IU/mL of factor VIIa andbetween 4 and 30 μmol/mL, more preferably between 8 and 20 μmol/mL ofthe calcium ion source.

In particular, the factor XIII is present in an amount from 30 IU/mL to700 IU/mL, preferably from 100 IU/mL to 400 IU/mL.

The concentrations and activities are per millilitre of final liquidbiological adhesive solution. Such a solution may notably be obtained byreconstitution of freeze-dried components. As indicated above, theseplasma factors preferably present in such concentrations provide thesought functionalities for the present biological adhesive.

Sources of calcium ions represent water-soluble components, compatiblewith clinical use; preferably these components are inorganic salts, suchas calcium chloride (CaCl₂) or calcium gluconate.

As indicated above, the biological adhesive of the invention is readyfor use and comprises a homogenous mixture of all the components inliquid form. It may also appear in frozen form, which makes it suitablefor extended storage in this form for at least 2 years without any riskof formation of fibrin, which might be observed after unfreezing. Simpleresetting to room temperature is sufficient so that the adhesive may beused.

The invention also relates to a thrombin-free fibrinogen-basedbiological adhesive for therapeutic use, comprising the factor VIIa anda source of calcium ions, as defined earlier, appearing in afreeze-dried form, suitable for extended storage. It may be obtained byapplying known freeze-drying techniques for the adhesive in the liquidform. The availability of such a presentation of the adhesive has thedecisive advantage of only requiring simple reconstitution of thefreeze-dried product comprising both active ingredients and the sourceof calcium ions in a biologically compatible solvent or aqueous mediumin order to obtain the stable liquid adhesive, this preparation beingcarried out beforehand anticipating its use. Further, such afreeze-dried adhesive may very easily be stored at room temperature forat least 2 years without any risk of formation of fibrin, which might beobserved after reconstitution. It may easily be transported right up toa site where a patient requires use of this adhesive.

The invention also relates to a kit for preparing the biologicaladhesive according to the invention comprising packaging meanscomprising a batch of freeze-dried fibrinogen plasmatic factor, a batchof freeze-dried FVIIa plasmatic factor, a batch of calcium ions sourceas a powder and an aqueous solvent.

Such a kit notably comprises three different individual batches in dryform, each in particular comprising one of the constitutive componentsof the adhesive of the invention, and in fact it is an intermediateproduct from which it will be then easy to merge and dissolve thebatches in a biologically compatible solvent or aqueous medium, such asinjectable purified water (PPI), for obtaining the stable liquidbiological adhesive of the invention, if need be, concentrated, whichmay then be frozen. The advantage of making such a kit available is thepossibility of extended storage of the three batches of differentcomponents for at least 2 years at room temperature, without observingformation of fibrin upon reconstitution, and obtaining the stable andliquid biological adhesive by simple dissolution.

In fact, the packaging means form means with which the components of theadhesive may be in particular stored and prepared, and the adhesive maybe dispensed before use. They further advantageously comprise at leastone container for at least three of the different components of theadhesive, including preferably at least one for each component. Eachcontainer is intended to receive one of the components, which is thendissolved in the aqueous solvent. The containers may be flasks invarious glass type materials and in biologically compatible polymers.

Preferably, the packaging means may advantageously be a single containercontaining at least three components. Such a packaging has the advantagethat a simple dissolution of the whole directly provides the liquidadhesive ready for use.

Advantageously, the kit also comprises a device for dispensing theliquid adhesive once it is prepared by dissolving the above batches withthe aqueous solvent. Such a device for example represents a syringe of asuitable volume, depending on the dose of adhesive to be delivered,including a fine needle, typically of a diameter less than 2 mm, inparticular less than 1 mm, or else a conventional catheter.

Preferably, the above kit includes a two-component batch from a mixtureof the batches of said freeze-dried plasma factors, and the calcium ionsource batch as a powder. This kit therefore comprises a batch of afreeze-dried mixture of the fibrinogen and FVIIa plasmatic factors ofthe invention on the one hand, and a batch of the calcium ion source asa powder on the other hand, capable of also being in the form of afreeze-dried product, for which it will be simply sufficient to mergethem together and mix them up with an aqueous medium such as PPI water,before use. Alternatively, the kit may comprise a batch of afreeze-dried mixture of plasmatic factors, and a calcium ion sourcebatch as an aqueous solution on the other hand.

Preferably, the kit of the invention is characterized by the fact thateach freeze-dried fibrinogen or FVIIa plasma factor batch or thetwo-component batch of the mixture of said batches of freeze-driedplasma factors comprises constituents of a pharmaceutically acceptablefreeze-drying stabilizing formulation. The same applies to thefreeze-dried biological adhesive.

Indeed, the different freeze-dried products of the plasmatic factors areobtained by freeze-drying liquid concentrates or solutions of theplasmatic factors or each of these factors, according to conventionallyapplied techniques, advantageously comprising for this purpose, afreeze-drying stabilizing formulation as described in Patent ApplicationFR 04 02001 filed by the Applicant. In this case, the stabilizingformulation advantageously represents a mixture of arginine, at leastone hydrophobic amino acid and trisodium citrate and may further beadded with glycine and/or lysine. Advantageously, the concentration ofeach of the additives per litre of protein concentrate is the following:

-   -   arginine from 25 to 50 g/L and preferably from 35 to 45 g/L        (with reference to U.S. Pat. No. 5,399,670);    -   trisodium citrate, from 0.5 to about 12 g/L;    -   leucine, isoleucine or their mixtures from 5 to 15 g/L and        preferably from 9 to 11 g/L; and    -   glycine and/or lysine, each from 1 to 5 g/L and preferably each        from 1.5 to 2.5 g/L.

The stabilizing formulation may also, if need be, comprise stabilizingadjuvants known in the art.

Consequently, the invention also relates to the use of the kit accordingto the invention for preparing a liquid but possibly frozen biologicaladhesive, by reconstitution of the three batches of said kit in abiologically compatible aqueous solvent, if necessary followed byfreezing.

The invention also relates to a biological adhesive as described earlierfor use as a medicament. In the case when the adhesive is stored in afrozen form, it will be sufficient to unfreeze the latter before use. Inthe case of a freeze-dried adhesive, reconstitution in a biologicallycompatible aqueous solvent allows it to be used for the sought effects.

The biological adhesive of the invention is therefore used for preparinga medicament intended for haemostasis and/or heeling of damagedbiological tissues, such as the skin or any organ capable of beingsurgically operated (spleen, liver, lungs, intestines, etc.), which asexplained earlier, may represent cruentous tissues or haemorrhagicwounds, the bleeding of which may even be very low insofar that all thefactors required for the coagulation cascade are present. The adhesivemay be used in the presence of plasma for preparing a medicamentintended to treat damaged tissues, for example for joining thesetissues, selected from the group formed by cartilage, collagen, bone andbone powder.

Further, in the presence of plasma, it is also used for preparing amedicament intended to merge biomaterials selected from the group formedby alginates of polylactic acid. The presence of plasma is consequentlyrequired in certain cases for exogenous supply of plasma factors inorder to initiate the coagulation cascade, and preferably it iscompatible or autologous plasma. This supply may notably be performedwithin the scope of conventional surgical operations in stomatology orodontology.

The biological tissue may also stem from cultured and differentiatedstem cells.

Although the adhesive of the invention is a medicament, in particular adressing for local application, i.e. for external use on a wound orother injury as described above, it is not excluded that the medicamentmay also be a suitable gelatine capsule, either gastro-resistant or not,in which the biological adhesive is in a dry form, and ingested fortreating digestive bleeding.

The stability of at least 24 hours of the adhesive of the inventionallows it to remain fluid and therefore be able to be used for preparinga medicament intended to embolize nutritive blood vessels of a tumoraltarget. This may advantageously be achieved by an injection route whichuses a conventional catheter right up to these tumoral targets in orderto thereby “dry” the tumor.

With the adhesive, haemostasis may be provided through an endoscopicsystem used in microsurgery (samplings, biopsies, excision of polyps,etc.).

The fluidity of the present adhesive, notably related to its stability,makes its use possible through a fine needle, typically of a diameterless than 1 mm, in particular less than 0.5 mm, for application insurgery under a microscope, for example an ophthalmic application.

The following example illustrates the invention without however limitingits scope.

EXAMPLE 2 mL of a biological adhesive sample of the invention (sample A)is prepared, comprising fibrinogen at a concentration of 80 mg/mL, 100IU/mL of factor VIIa and 5 μmol/mL of calcium chloride which areintroduced into a single syringe.

A standard biological adhesive sample B of the prior art is alsoprepared, comprising a mixture B1 consisting of fibrinogen at aconcentration of 80 mg/mL and containing 100 IU/mL of factor XIII on theone hand, and a mixture B2 consisting of calcium thrombin at 500 IU/mLon the other hand. The respective mixtures B1 and B2 are introduced intotwo distinct syringes, the ends of which are arranged so as to include asingle needle thereby allowing the mixtures B1 and B2 to be mergedtogether.

An anaesthetized rabbit is submitted to laparotomy. The liver is exposedand one proceeds with a first incision of the organ in order to causebleeding on this section. After dabbing the excess of blood with acompress, the biological adhesive sample B is immediately applied on theincised section by simultaneous expulsion of the contents of bothsyringes by the single needle which allows a homogenous mixture of B1and B2 to be prepared. The banks of the wound are kept joined for 30seconds in order to let the biological adhesive set.

After heeling, it is observed that the obtained scar has irregularitiesand “lumps” of visible fibrin. These clusters are due to inhomogeneoussetting, probably because of the fibrin formed before adhesion.

A second incision is performed at another location of the liver which issurgically treated in the same way as previously. The biologicaladhesive sample A according to the invention, contained in a singlesyringe, is applied on the incised section and the banks of the woundare kept joined for 35 seconds in order to let the biological adhesiveset.

After heeling, it is observed that the obtained scar has a clear lineand withstands any deshience attempt. No trace of excess fibrin isvisible, which shows that the excess adhesive has not coagulated in theabsence of an operating wound and therefore without the presence oftissue factors.

1. A thrombin-free, prothrombin-free, fibrinogen-based stablesingle-compound liquid biological adhesive for therapeutic use,comprising a solvent and a mixture of fibrinogen, factor VIIa, andcalcium ions.
 2. The biological adhesive according to claim 1, whereinthe solvent is water.
 3. The biological adhesive according to claim 1,wherein the fibrinogen has a fibrinogen content between 60 and 120 mg/mLof final liquid biological adhesive solution.
 4. The biological adhesiveaccording to claim 3, wherein the fibrinogen content is from 80 to 100mg/mL of the final liquid biological adhesive solution.
 5. Thebiological adhesive according to claim 1 wherein the factor VIIa has anactivated factor VII content from 50 to 500 IU of activated factor VIIper millilitre of final liquid biological adhesive solution (50-500IU/mL).
 6. The biological adhesive according to claim 5, wherein theactivated factor VII content is from 70 to 300 IU/mL of activated factorVII, in particular from 80 to 120 IU/mL.
 7. The biological adhesiveaccording to claim 1, wherein the calcium ions are in a concentrationcomprised between 4 and 30 μmols/mL of final liquid biological adhesivesolution.
 8. The biological adhesive according to claim 7, wherein thecalcium ions are in a concentration comprised between 8 and 20 μmols/mL.9. The biological adhesive according to claims 1, further comprising thefactor XIII.
 10. The biological adhesive according to claim 9, whereinthe factor XIII is present in an amount from 100 IU to 400 IU permillilitre of final liquid biological adhesive solution (100 IU/mL-400IU/mL).
 11. The biological adhesive according to claim 1, to 10appearing in a frozen form, suitable for extended storage.
 12. Thebiological adhesive according to claim 1, in a freeze-dried form,suitable for extended storage.
 13. The biological adhesive according toclaim 12, further comprising constituents of a pharmaceuticallyacceptable freeze-drying stabilizing formulation, preferablyrepresenting a mixture of arginine, at least one hydrophobic amino acidand trisodium citrate. 14.-29. (canceled)